Factor involved in latent infection with herpesvirus, and use thereof

ABSTRACT

Disclosed are a protein and a gene each of which is a factor involved in latent infection with a herpesvirus. An antibody against the factor was detected in approximately 50% of patients suffering from mental disorders, whereas the antibody was hardly detected in healthy persons. Further, a mouse having SITH-1 introduced therein developed a mental disorder such as a manic-depressive illness or depression-like disorder. Based on these findings, it is possible to provide a method for objectively determining a mental disorder and an animal model of a mental disorder.

This application is a Divisional of copending application Ser. No.12/679,816 filed on Mar. 24, 2010 and for which priority is claimedunder 35 U.S.C. §120, which is a National Phase of PCT InternationalApplication No PCT/JP2008/067300, filed on Sep. 25, 2008, under 35U.S.C. §371, which claims priority under 35 U.S.C. 119(a) to PatentApplication No. JP 2007-250461, filed on Sep. 27, 2007. The entirecontents of all of the above applications are hereby expresslyincorporated by reference into the present application.

TECHNICAL FIELD

The present invention relates to: a factor involved in latent infectionwith a herpesvirus; and use thereof. Particularly, the present inventionrelates to a novel protein which is specifically expressed during latentinfection with a herpesvirus; a gene encoding said protein; and usethereof.

BACKGROUND ART

Viruses of the family Herpesviridae, each having an overall size ofapproximately 150 nm to 200 nm, are such that a core protein issurrounded by multi-stranded DNA with molecular masses of 80 to 150×10⁶daltons. This multi-stranded DNA is enclosed in an icosahedral capsidwhich has a diameter of approximately 100 nm and is made of 162capsomers, so as to form a nucleocapsid which is surrounded by anenvelope. Herpes viruses have been found in almost all mammals andamphibians. In particular, viruses of the family Herpesviridae that havehost specificity for humans are named human herpesviruses (HHVs). HHVsare classified into subfamilies Alphaherpesvirinae (e.g., herpes simplexvirus and varicella-zoster herpes virus), Betaherpesvirinae (e.g.,cytomegalovirus), and Gammaherpesvirinae (e.g. EB virus).

These herpes viruses are characterized by having a stage of latentinfection. The “latent infection” refers to such a state of infectionthat a virus that has infected a host cell does not produce infectiousvirions within the host cell but continues to survive. Even in thisphase of latent infection, virus genes and gene products that help thevirus genes to exist are retained within the host cell. Herpes virusesthat exhibit latent infection are known to resume production of virionsand viral replication in a large amount owing to certain causes on theside of the host (e.g. growing old and somatic complaints (includingfatigue)). This state is called “reactivation”.

In short, herpes viruses have the following unique character: Herpesviruses continue to infect the host latently as long as the host hasnothing abnormal; however, once a somatic disturbance occurs in the hostand the viruses detect that the host is in danger, the viruses arereactivated to seek another, healthy host.

To study the biology of such viruses of the family Herpesviridae,understanding their latent infection and reactivation is essential.However, among the many herpes viruses, it is only EB virus belonging tothe subfamily Gammaherpesvirinae that has been studied to yield manyfindings about latent infection, and much remains unclear about otherviruses.

In particular, concerning factors that may be involved in latentinfection of Betaherpesvirinae, there has been obtained no informationother than from the findings previously made by the present inventors.For example, Non-Patent Document 1 discloses latent infection of HHV-6in macrophages in peripheral blood which macrophages have differentiatedto a relatively high extent, and identifies the sites in a host at whichsites the host is latently infected with HHV-6. Non-Patent Document 2describes very frequent invasion of HHV-6 into a brain upon primaryinfection to cause persistent infection and latent infection. Non-PatentDocument 3 discloses genes (latent infection genes) that are expressedduring latent infection of HHV-6, and suggests that those genes play therole of regulating latent infection and reactivation of the virus.

Non-Patent Document 4 shows that the state of latent infection withHHV-6 involves an intermediate stage which is comparatively stable andallows for active gene expression, with the result that a latentinfection gene and a protein (latent infection gene protein) encoded bythis gene are expressed abundantly. What is more, Non-Patent Document 5shows that patients with chronic fatigue syndrome had in their seraantibodies against latent infection gene proteins which are expressed atan increased level in the intermediate stage.

-   Non-Patent Document 1-   Kondo. K et al. Ltatent human herpesvirus 6 infection of human    monocytes/macrophages (J Gen Virol 72:1401-1408, 1991)-   Non-Patent Document 2-   Kondo. K et al. Association of human herpesvirus 6 infection of the    central nervous system with recurrence of febrile convulsions. (J    Infect Dis 167:1197-1200, 1993.)-   Non-Patent Document 3-   Kondo. K et al. Identification of human herpesvirus 6    latency-associated transcripts. (J. Virol. 76: 4145-4151, 2002)-   Non-Patent Document 4-   Kondo K et al. Recognition of a Novel Stage of Beta-Herpesvirus    Latency in Human Herpesvirus 6. (J. Virol. 77: 2258-2264, 2003)-   Non-Patent Document 5-   Kazuhiro Kondo, “Herpesvirus Kansen to Hiro (Herpesvirus latency and    fatigue)”, Virus, 2005, Vol. 55, No. 1, pages 9 to 18

SUMMARY OF INVENTION

However, there has not been identified any latent infection gene orlatent infection gene protein specifically involved in diseases. Inaddition, its functions and a relationship with a pathogenic mechanismof chronic fatigue syndrome have remained unknown. Further, there is apossibility that HHV-6 is involved in other diseases in addition tochronic fatigue syndrome.

Therefore, it has been strongly demanded to make clear the relationshipbetween infection with HHV-6 and diseases, and also to develop atechnique contributing to establishment of (i) an objective diagnosismethod for diseases and (ii) an animal model.

The present invention was made in view of the foregoing problems, and anobject of the present invention is to identify a factor involved inlatent infection with HHV-6 and to provide use thereof.

In order to solve the foregoing problems, the present inventors made adiligent study. As a result, the present inventors reached the followingunique idea: In light of the HHV-6's distinctive nature, i.e., thelatent infection and the reactivation, identifying a factor involved inthe latent infection and the reactivation would yield a finding aboutthe relationship between infection with HHV-6 and mental disorders.Based on this idea, the present inventors conducted complicated,sophisticated experiences many times. As a result, the present inventorsidentified: a novel gene expressed at the intermediate stage, at which agene specific for latent infection with HHV-6 is expressed actively; anda novel protein (Small protein encoded by the Intermediate Transcript ofHHV-6-1; SITH-1) encoded by the novel gene. Further, the presentinventors conducted functional analysis of the novel gene and theprotein SITH-1, which is encoded by the novel gene, so as to make thefollowing new findings: (i) the protein SITH-1 has ability to increasean intracellular calcium concentration; and (ii) an antibody against theprotein SITH-1 is significantly detected in patients with mooddisorders, but is hardly detectable in healthy persons. Thus, thepresent invention was completed. The present invention was completedbased on the above new findings, and includes the following inventions:

(1) A gene encoding:

(a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or

(b) a protein having an amino acid sequence with a substitution,deletion, insertion, and/or addition of one or several amino acids inthe amino acid sequence shown in SEQ ID NO: 1, the protein havingactivity of increasing an intracellular calcium concentration.

(2) A gene including an open reading frame region having the nucleotidesequence shown in SEQ ID NO: 2.

(3) A gene encoding a protein that hybridizes under stringenthybridization conditions with DNA having a nucleotide sequencecomplementary to DNA having the nucleotide sequence shown in SEQ ID NO:2 or 3, the protein having activity of increasing an intracellularcalcium concentration.

(4) A protein encoded by a gene as set forth in any one of (1) through(3).

(5) A protein being:

(a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or

(b) a protein having an amino acid sequence with a substitution,deletion, insertion, and/or addition of one or several amino acids inthe amino acid sequence shown in SEQ ID NO: 1, the protein havingactivity of increasing an intracellular calcium concentration.

(6) An antibody that recognizes a protein as set forth in (4) or (5).

(7) A recombinant expression vector including a gene as set forth in anyone of (1) through (3).

(8) A transformant which is produced by transfer of a gene as set forthin any one of (1) through (3) or a recombinant expression vector as setforth in (7).

(9) A gene detection instrument including a probe having at least partof a nucleotide sequence, or its complementary sequence, of a gene asset forth in any one of (1) through (3).

(10) A detection instrument including a probe which is a polypeptidehaving at least part of an amino acid sequence of a protein as set forthin (4) or (5).

(11) A determination method including the step of: determining whetheror not an antibody as set forth in (6) exists in a subject.

(12) The determination method as set forth in (11), wherein: thedetermining is made by means of immunological detection through use of aprotein as set forth in (4) or (5) or a partial fragment of the protein.

(13) The determination method as set forth in (11) or (12), wherein: thedetermining is made by using a biological sample isolated from thesubject.

(14) A determination kit for performing a determination method as setforth in any one of (11) through (13).

(15) The determination kit as set forth in (14), including at least oneselected from:

(i) a protein as set forth in (4) or (5);

(ii) a partial fragment of the protein (i); and

(iii) an instrument to which the protein (i) or the partial fragment(ii) is immobilized.

(16) A diagnosis method for diagnosing whether or not a human subjecthas a mental disorder, including the steps of: (i) determining whetheror not an antibody as set forth in (6) exists in a human subject,according to a determination method as set forth in any one of (11)through (13); and (ii) determining that the human subject contractschronic fatigue syndrome, in a case where the step (i) determines thatthe antibody as set forth in (6) exists in the human subject.

(17) The diagnosis method as set forth in (16), wherein: in the step(i), the determining is made by using a biological sample isolated fromthe human subject.

(18) A diagnosis method for diagnosing whether or not an animal subjecthas a mental disorder, including the steps of: (i) determining whetheror not an antibody as set forth in (6) exists in an animal subject,according to a determination method as set forth in any one of (11)through (13); and (ii) determining that the animal subject has a mentaldisorder, in a case where the step (i) determines that the antibody asset forth in (6) exists in the animal subject.

(19) A diagnosis kit for performing a diagnosis method as set forth inany one of (16) through (18).

(20) The diagnosis kit as set forth in (19), including at least oneselected from:

(i) a protein as set forth in (4) or (5);

(ii) a partial fragment of the protein (i); and

(iii) a detection instrument to which the protein (i) or the partialfragment (ii) is immobilized.

(21) An animal model determination method for determining whether or notan animal subject is useful as an animal model of a mental disorder,including the steps of: (i) diagnosing whether or not an animal subjecthas a mental disorder, according to a diagnosis method as set forth in(18); and (ii) determining that the animal subject is useful as ananimal model of the mental disorder, in a case where the step (i)diagnoses that the animal subject has the mental disorder.

(22) An animal model produced by transfer of a gene as set forth in anyone of (1) through (3), a gene product thereof, or a recombinantexpression vector as set forth in (7).

(23) A screening method for performing screening for a candidatesubstance for a psychotropic agent, including the steps of: (i)administering, to an animal model of a mental disorder, a subjectsubstance; (ii) determining whether or not the mental disorder of theanimal model is cured or improved, according to a diagnosis method asset forth in (18); and (iii) determining that the subject substance is acandidate substance for a psychotropic agent, in a case where the metaldisorder of the animal model is determined to be cured or improved.

It is more preferable that the screening method (23) is performed bothby (i) the diagnosis method (18) and (ii) a diagnosis method using e.g.,a (heretofore known) behavior disorder and/or startle response of ananimal.

A gene or a protein of the present invention is expressed specificallyduring latent infection with a herpesvirus, and has ability to regulatelatent infection and reactivation of a herpesvirus. Further, asdescribed later, it has been shown that an antibody against a protein ofthe present invention is significantly found in patients with mentaldisorders. Therefore, determining the presence or absence of theantibody enables objective diagnosis for mental disorders.

Furthermore, a gene or a protein of the present invention is applicableto diagnosis for various diseases, as well as diagnosis for the diseasesdescribed herein. Moreover, a gene or a protein of the present inventionis also available for use in drug screening methods, animal modelproducing methods, and various kinds of kits, for example.

For a fuller understanding of the nature and advantages of theinvention, reference should be made to the ensuing detailed descriptiontaken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing schematically a structure of a latentinfection specific gene and positions of analytical primers.

FIG. 2 is a diagram showing the results of amplification performed bythe PCR technique with respect to HHV-6 gene products.

FIG. 3 is a diagram showing the results of analysis performed by theRACE technique with respect to novel latent infection specific genemRNAs.

FIG. 4 is a diagram showing the results of an experiment in which a hostprotein binding to the protein SITH-1 was identified by the yeasttwo-hybrid assay.

FIG. 5 is a diagram showing that a protein SITH-1 increased an amount ofCAML in an astrocyte-like glial cell line.

FIG. 6 is a diagram showing how SITH-1 increased a calcium concentrationin glial cells.

FIG. 7 is a graph showing antibody titers to SITH-1 in patients withmental disorders.

FIG. 8 is a graph showing the result of investigating an effect ofSITH-1 in a tail suspension test.

FIG. 9 is a graph showing the result of investigating an effect of in aforced swimming test.

FIG. 10 is a graph showing the result of investigating an effect ofSITH-1 in terms of startle response (prepulse inhibition).

FIG. 11 is a graph showing the result of an experiment in which SITH-1was expressed in mouse glial cells using an adenovirus vector and, threeweeks later, the animals were measured for their motor activity underwheel running activity.

FIG. 12 is a graph showing the result of an experiment in which SITH-1was expressed in mouse glial cells using a lentivirus vector and, eightweeks later, the animals were measured for motor activity under wheelrunning activity.

FIG. 13 is a graph showing the results of diagnosing, with SITH-1 usedas a marker, various diseases that are complicated by depression.

DESCRIPTION OF EMBODIMENTS

The following describes one embodiment of the present invention.However, the present invention is not limited to this.

First, to help understanding of the present invention, how the presentinventors completed the present invention will be described briefly. Thepresent inventors speculated that infection with HHV-6 among the variouskinds of human herpes viruses was most probably a cause of mentaldisorders, particularly ones accompanied by mood disorders. The reasonsinclude: (i) among symptoms of chronic fatigue syndrome (CFS) for whichHHV-6 has heretofore been held as one cause, depressive symptoms andothers that are often found in mental disorders are recognized; (ii)HHV-6 causes latent infection in a brain; and (iii) an antibody reactivewith the heretofore identified HHV-6 latent infection specific geneprotein, as well as an antibody reactive with an unknown protein thatwas expressed in cells latently infected with HHV-6 but which was yet tobe identified for a gene or for itself were detected at high frequenciesin the sera of CFS patients.

Further, in light of the fact that the primary sites in a brain whichare latently infected with HHV-6 include a frontal lobe and ahippocampal region each of which governs human thoughts and emotions, aswell as the fact that viruses causing latent infection in a brain arejust a few including HHV-6, the present inventors speculated therelationship between HHV-6 and mental disorders. Furthermore, HHV-6 isknown to cause latent infection in glial cells (e.g., astrocytes) thatplay important roles in metabolism of substances within a brain (e.g.,serotonin) that are associated with depression. Also in terms of this,the present inventors reached the unique idea that HHV-6 might beassociated with mental disorders such as mood disorders.

Thus, the present inventors speculated that patients with CFS mightinclude considerable cases who present with psychiatric symptoms onaccount of the latent infection of the brain with HHV-6. In particular,the present inventors speculated the relationship between HHV-6 and mooddisorders such as depression and manic-depressive illness.

Mood disorders are symptoms found in mental disorders such as depressionand manic-depressive illness, and two most typical examples aredepression that presents with only symptoms of depression andmanic-depressive illness in which episodes of mania alternate withepisodes of depression. While various possible causes have beenproposed, including stress, genetic aberrations, and infection, nosingle factor has yet been established. The incidence of mood disordersis increasing these days, and this is becoming a big social problem.Therefore, it is desirable that the etiology and pathology of each mooddisorder are unraveled and methods for diagnosing and treating it aredeveloped as soon as possible. A problem worth particular mention hereis that diagnosis of mood disorders is liable to be only qualitative,and involves difficulty in achieving objectivity. In addition, animalmodels contributing to studies of mood disorders and development ofmethods for treating them have not been developed adequately. Thishinders clarification of the etiology and development of the treatingmethods.

On this account, the present inventors thought that it necessary to makeclear the relationship between (i) infection with HHV-6 and (ii) mooddisorders and mental disorders, and to develop a technique contributingto establishment of objective diagnosis and animal models for mooddisorders and mental disorders.

Needless to say, these speculations are unique ones at which the presentinventors arrived as a result of a diligent study made in this researchfield for a long time, and cannot be easily arrived at by a generalperson skilled in the art.

The following describes details of a protein, a gene, and others of thepresent invention in order.

(1) Protein and Gene of the Present Invention

(1-1) Structure

The present invention provides a factor that is involved in latentinfection with a herpes virus. In greater detail, the present inventionprovides (i) a protein that is expressed specifically during latentinfection with a herpes virus and (ii) a gene encoding the protein. Thephrase reading “expressed specifically during latent infection with aherpes virus” means that a gene derived from a herpes virus or a geneproduct thereof is expressed specifically in a virally infected hostwhile the host is latently infected (but not productively infected) withthe herpes virus.

The protein and the gene may be, for example, (a) a protein having theamino acid sequence shown in SEQ ID NO: 1 and a gene encoding theprotein.

As will be described later in the Example, the protein having the aminoacid sequence shown in SEQ ID NO: 1 is isolated and identified as aprotein that is expressed specifically during latent infection withhuman herpesvirus-6 (HHV-6). This protein is hereinafter referred to as“Small protein encoded by the Intermediate Transcript of HHV-6-1(protein SITH-1)”. The protein SITH-1 is a protein which has a molecularmass of approximately 17.5 kDa, the amino acid sequence shown in SEQ IDNO: 1, and 159 amino acids.

The protein SITH-1 is encoded by an SITH-1 gene. As shown in SEQ ID NO:3, cDNA of the SITH-1 gene has a size of 1795 base pairs (approximately1.79 kbp). Further, the 954^(th) to 956^(th) nucleotide sequencerepresents a start codon (Kozak ATG), whereas the 1431^(st) to 1433^(rd)nucleotide sequence represents a stop codon (TAA). Hence, the SITH-1gene has an open reading frame (ORF) having the 954^(th) to 1430^(th)nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 3,with the ORF having a size of 477 base pairs (approximately 0.48 kbp).The nucleotide sequence that represents the ORF region of the cDNA ofSITH-1 is shown in SEQ ID NO: 2. Note that the nucleotide sequence shownin SEQ ID NO: 2 includes three bases of the stop codon.

The protein of the present invention may be, for example, (b) a proteinhaving an amino acid sequence with a substitution, deletion, insertion,and/or addition of one or several amino acids in the amino acid sequenceshown in SEQ ID NO: 1, the protein being expressed specifically duringlatent infection with a herpes virus. The gene of the present inventionmay be, for example, a gene encoding this protein.

The phrase reading “with a substitution, deletion, insertion, and/oraddition of one or several amino acids” means substitution, deletion,insertion, and/or addition of numbers of amino acids (for example,preferably 10 or less, more preferably 7 or less, further preferably 5or less) that can be brought about by a known mutant peptide producingmethod such as site-directed mutagenesis. Thus, the protein (b) may bedescribed as being a mutant protein of the protein (a). Note that the“mutant” herein primarily refers to a mutant made by artificialintroduction by means of a known mutant protein producing method, or maybe one obtained by isolation and purification of a naturally-existing,similar mutant protein.

Alternatively, the gene of the present invention may be, for example, agene encoding (c) a protein that hybridizes under stringenthybridization conditions with DNA having a nucleotide sequencecomplementary to DNA having the nucleotide sequence shown in SEQ ID NO:2, the protein being expressed specifically during latent infection witha herpes virus.

The phrase reading “hybridizes under stringent hybridization conditions”means that hybridization occurs only in a case where nucleotidesequences of interest have at least 90% identity, preferably at least95% identity, most preferably at least 97% identity. As a specificexample of the “stringent hybridization conditions”, the followingcondition is possible: A hybridization filter is incubated overnight at42° C. in a hybridization solution (including 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA), followed by being washed in 0.1×SSC atapproximately 65° C. Further, the hybridization can be performed by aconventionally-known method, for example, according to the proceduresdescribed in “J. Sambrook et al. Molecular Cloning, A Laboratory Manual,2d Ed., Cold Spring Harbor Laboratory (1989)”, and is not limited to anyspecific one. Generally, as the temperature rises and the saltconcentration becomes lower, the level of the stringency increases(i.e., more difficult to hybridize).

Note that the term “gene” herein is used interchangeably with“polynucleotide”, “nucleic acid” or “nucleic acid molecule”. The“polynucleotide” refers to a polymer of nucleotides. Thus, the term“gene” herein includes not only double-stranded DNA but alsosingle-stranded DNAs (e.g., a sense strand and an antisense strandconstituting the double-stranded DNA) and RNA (e.g., mRNA). Theantisense strand may be used as a probe or an antisense drug. The term“DNA” includes e.g., (i) cDNA obtained by cloning, a chemical synthesistechnique, or a combination thereof and (ii) a genomic DNA. That is, the“DNA” may be a “genomic” DNA including a noncoding sequence (e.g.,intron), which genomic DNA is a form contained in animal genomes.Alternatively, the “DNA” may be cDNA obtained from mRNA by using reversetranscriptase or polymerase, i.e., “transcriptional” DNA including nononcoding sequence (e.g., intron). Further, the gene of the presentinvention may be one having not only a sequence encoding the amino aciddescribed concerning the above (a) or (b) but also a sequence of anuntranslated region (UTR) and/or a vector sequence (including anexpression vector sequence). Further, the mRNA or the cDNA may include,at an end and/or the inside of its translated region, a desiredpolynucleotide such as a regulatory sequence or a polyadenylic acidsequence. Furthermore, in a case where the protein of the presentinvention can be encoded by a plurality of alleles, the term “nucleicacid” encompasses all of the alleles, their transcripts, and cDNA. Notethat the term “nucleic acid” herein includes a polynucleotide includingdesired simple nucleotides and/or modified nucleotides, examples ofwhich encompass cDNA, mRNA, total RNA, and hnRNA. The term “modifiednucleotides” encompasses: phosphate esters such as inosine,acetylcytidine, methylcytidine, methyladenosine, and methylguanosine;and nucleotides that can be acquired by an effect of ultraviolet rays orchemical substances.

The term “nucleotide sequence” is used interchangeably with “nucleicacid sequence”, and is presented as a sequence of deoxyribonucleotides(each abbreviated as A, G, C, or T). Further, a polynucleotide or a“nucleotide sequence” of a polynucleotide is intended to mean (i) asequence of deoxyribonucleotides for a DNA molecule or a polynucleotideand (ii) a sequence of ribonucleotides (A, G, C, and U) (each thymidine(T), which is a deoxynucleotide, in the deoxynucleotide sequencespecified herein is replaced with uridine (U), which is aribonucleotide) for an RNA molecule or a polynucleotide.

For example, an RNA molecule having the sequence shown in SEQ ID NO: 2or 4, which is represented by abbreviations for deoxyribonucleotides, isintended to mean an RNA molecule having a sequence in whichdeoxynucleotides A, G, and C shown in SEQ ID NO: 2 or are substitutedwith their corresponding ribonucleotides A, G, and C and deoxynucleotideT shown in SEQ ID NO: 2 or 4 is substituted with ribonucleotide U.Further, a polynucleotide having the nucleotide sequence shown in SEQ IDNO: 2 or 4 or a fragment of the polynucleotide is intended to mean apolynucleotide having a sequence represented by deoxynucleotides A, G, Cand/or T shown in SEQ ID NO: 2 or 4 or a fragment of the polynucleotide.

A fragment (partial sequence) of the gene of the present invention maybe used as a primer for polymerase chain reaction (PCR) or as ahybridization probe. The fragment (polynucleotide) is available inspecific PCR amplification of a homologue or an orthologue of the geneof the present invention, and is also available as a hybridization probewhich specifically hybridizes with a homologue or an orthologue of thegene of the present invention. That is, in a preferable embodiment, thefragment of the gene of the present invention is useful for diagnosis(i) as a primer for amplification of a target sequence performed bymeans of polymerase chain reaction (PCR) or (ii) as a probe according toa conventional DNA hybridization technique.

Further, other examples for use of the fragment of the gene of thepresent invention encompass: in situ hybridization (e.g., FISH) withrespect to a mitotic chromosome spread, by which in situ hybridization acorrect chromosome site is shown (described in Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988)); and northern blotting analysis for detection of mRNA of thepresent invention expressed in a certain tissue.

Examples of the gene of the present invention encompass, but are notlimited to: a polynucleotide by itself encoding an amino acid sequenceof a maturation protein; a coding sequence of a maturation protein andits further sequence (e.g., a sequence encoding a leader sequence)(e.g., a preprotein sequence, a proprotein sequence or a preproproteinsequence); intron, a non-coding 5′ sequence and a non-coding 3′ sequence(e.g., a transcription untranslated region working in transcription andmRNA processing (including splicing and a polyadenylated signal)); and afurther coding sequence encoding another amino acid providing furtherfunctionality.

Therefore, for example, a sequence encoding a protein may be fused to amarker sequence (e.g., a sequence encoding a peptide which facilitatespurification of a fused protein). In a preferable embodiment of thepresent invention, a marker amino acid sequence may be a hexa-histidinepeptide, such as a tag provided in pQE vector (Qiagen, Inc.). Asdescribed in “Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824(1989)”, the hexa-histidine peptide is useful in purifying a fusionprotein in a simple manner. Alternatively, a publicly and/orcommercially available marker amino acid sequence of many kinds can beused. For example, as described in “Wilson et al., Cell 37: 767 (1984)”,an “HA” tag is another peptide useful in purification, which HA tagcorresponds to an epitope derived from an influenza hemagglutini (HA)protein. Further alternatively, a fusion protein made by causing the Fcto be fused with the N-terminal or the C-terminal of the protein of thepresent invention would be useful for purification.

Further, the present invention encompasses a mutant of the gene of thepresent invention. The mutant can naturally occur as well as a naturalallele mutant does. The “allele mutant” is intended to mean one of someinterchangeable forms of a gene occupying a predetermined gene locus ona chromosome of an organism. Further, a non-naturally-occurring mutantmay be produced by using e.g., a mutagenesis technique known in the art.Examples of such the mutant encompass a mutant produced by asubstitution, deletion, or addition of one or several nucleotides, asdescribed above. The substitution, deletion, or addition may occur atone or more nucleotides. The mutant may include mutation occurred in acoding region, a non-coding region or both of them. Mutation in a codingregion may cause a conservative or nonconservative substitution,deletion, or addition of an amino acid.

In addition to the maturation protein, examples of a preferable proteinof the present invention encompass: an extracellular domain, atransmembrane domain, an intracellular domain, and a protein which lacksthe whole of or part of a transmembrane domain but includesextracellular and intracellular domains. The term “protein” herein isinterchangeably used with “polypeptide” or “peptide”. Further, thepresent invention provides a polypeptide with a substitution, addition,and/or deletion of one or several amino acids of a protein encoded bythe nucleotide sequence shown in SEQ ID NO: 2. A conservative ornonconservative substitution, deletion, and/or addition of an aminoacid(s) is/are preferable, and a silent substitution, addition, and/ordeletion thereof is/are particularly preferable. These do not changecharacteristics and activity of the protein of the present invention orpart of the protein. In terms of this point, particularly preferable oneis a conservative substitution.

Further, the protein of the present invention may be not only the oneisolated from natural sources but also the one chemically synthesized orobtained by recombination. That is, the protein of the present inventionmay be isolated and purified from e.g., cells or tissues. Alternatively,the protein of the present invention may be expressed intracellularly bybeing encoded by a gene that has been transferred into the host cell.Further, the protein of the present invention may include an additionalpolypeptide.

The present invention relates to a polypeptide having an amino acidsequence of an epitope-bearing part of the protein described herein. Thepolypeptide having the amino acid sequence of the epitope-bearing partof the protein of the present invention only needs to include part of apolypeptide which part includes at least 6, 7, 8, 9 or 10 amino acids.In addition, such the polypeptide may also be an epitope-bearing-partpolypeptide having a length (optionally set) equal to or shorter than alength of an entire amino acid sequence of (i) a protein encoded by thenucleotide sequence shown in SEQ ID NO: 2 or 4 or (ii) a protein havingthe amino acid sequence shown in SEQ ID NO: 1.

In other words, the present invention provides an epitope-bearingpeptide of the protein of the present invention. As described in thelater-described Example, the protein of the present invention isimmunogenic. Therefore, it is possible to identify, in the protein ofthe present invention, an epitope part inducing an antibody response,according to a method known in the art. For example, Geysen, H. M. etal., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1984) discloses aprocedure of a rapid concurrent synthesis on solid supports of hundredsof peptides having sufficient purity to react in an enzyme-linkedimmunosorbent assay. Interaction of synthesized peptides with antibodiesis then easily detected without removing them from the supports. In thismanner, a peptide bearing an immunogenic epitope of a desired proteinmay be identified routinely by a person skilled in the art. For example,an immunologically important epitope in a coat protein of foot-and-mouthdisease virus was located by Geysen et al. with the resolution of sevenamino acids by the synthesis of an overlapping set of all 208 possiblehexapeptides covering an entire 213 amino acid sequence of a protein.Then, a complete replacement set of peptides in which all 20 amino acidswere substituted in turn at every position within the epitope wassynthesized, and particular amino acids conferring specificity for areaction with an antibody were determined. Thus, a peptide analog of theepitope-bearing peptide of the present invention can be made routinelyby this method. U.S. Pat. No. 4,708,781, Geysen (1987) describes furtherdetails of this method by which a peptide bearing an immunogenic epitopeof a desired protein is identified.

The “immunogenic epitope” is defined as part of a protein which partinduces an antibody response, in a case where the whole of the proteinis an immunogen. The immunogenic epitopes are considered to be limitedto two or more regions on a molecule. On the other hand, a site of aprotein molecule to which site an antibody can bind is defined as“antigenic epitope”. Generally, in a protein, the number of immunogenicepitopes is less than that of antigenic epitopes. For example, seeGeysen, H. M. et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1984).

An antigenic epitope-bearing peptide of the present invention is usefulfor induction of antibodies including a monoclonal antibody whichspecifically binds to the protein of the present invention. Therefore,most of hybridomas obtained by fusion of spleen cells taken from a donorimmunized with the antigenic epitope-bearing peptide secretes antibodiesgenerally reactive with natural proteins. The antibodies induced by theantigenic epitope-bearing peptide are useful to detect mimickedproteins, and antibodies against different peptides may be used fortracking the fate of various regions of a protein precursor whichundergo post-translational processing. A peptide and an anti-peptideantibody may be used in a variety of qualitative or quantitative assaysfor the mimicked proteins (e.g., in competition assays), since it hasbeen shown that, in immunoprecipitation assays, even short peptides(e.g., approximately 9 amino acids) can bind and substitute longerpeptides. For example, see Wilson, I. A. et al., Cell 37: 767-778 (1984)777. An anti-protein antibody of the present invention is also usefulfor purification of the mimicked proteins (e.g., by adsorptionchromatography using a method known in the art).

The antigenic epitope-bearing peptide of the present invention designedaccording to the above guideline preferably includes a sequence of atleast seven, more preferably of at least nine, most preferably betweenapproximately 15 to approximately 30 amino acids included in the aminoacid sequence of the protein of the present invention. However, apeptide or a polypeptide including a larger portion of the amino acidsequence of the protein of the present invention, containingapproximately 30 to approximately 50 amino acids, or any length up toand including the entire amino acid sequence of the protein of thepresent invention, also are considered the epitope-bearing peptide ofthe present invention, and also are useful for inducing antibodies thatreact with a mimicked protein. Preferably, an amino acid sequence of theepitope-bearing peptide is selected so that it can provide a substantialsolubility in an aqueous solvent (i.e., the selected sequence contains arelatively hydrophilic residue, and a highly-hydrophobic sequence ispreferably avoided); and a sequence containing a proline residue isparticularly preferable.

The epitope-bearing peptide of the present invention may be produced bydesired, conventional recombinant protein producing means which uses thegene of the present invention. For example, a short epitope-bearingamino acid sequence may be fused with a larger polypeptide which acts asa carrier, during production and purification of a recombinant andimmunization for producing an anti-protein antibody. The epitope-bearingpeptide may also be synthesized by using a known method for a chemicalsynthesis.

Further, the present invention may encompass a protein to be expressedinto which protein an appropriate secretory signal has beenincorporated, for secretion of a translated protein to the inside of alumen of an endoplasmic recticulum, to the inside of a periplasm space,or to an extracellular environment. The secretion signal may beendogenous with respect to a polypeptide, or may be a heterogenoussignal.

Therefore, the protein of the present invention can be expressed in amodified form such as a fusion protein, and may include not only thesecretion signal but also an additional heterogenous functional region.For example, an additional amino acid, particularly, a region of anelectrically charged amino acid can be added to the N-terminal of aprotein for improvement in stability and durability in the host cellsduring purification or subsequent manipulation and storage. Further, apeptide portion can be added to a protein for facilitating purification.Such a region can be removed before a final preparation of the protein.In particular, addition of a peptide portion to a protein for thepurpose of causing secretion or excretion, improving stability, andfacilitating purification is well known in the art, and is a techniqueroutinely performed.

A preferable fusion protein includes a heterogeneous region derived fromimmunoglobulin which heterogeneous region is useful for making a proteinsoluble. For example, EP A 0 464 533 (Canadian counterpart application2045869) discloses fusion proteins including various portions ofconstant regions of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, employing the Fc region of afusion protein is sufficiently advantageous for use in therapy anddiagnosis, thereby resulting in, for example, improved pharmacokineticproperties (EP A 0232 262). On the other hand, for some uses, it isdesirable that the Fc part is deleted after the fusion protein has beenexpressed, detected, and purified in an advantageous manner described.This is a case where the Fc portion proves to be a hindrance to use intherapy and diagnosis (e.g., in a case where the fusion protein is to beused as an antigen for immunizations). In drug screening, for example,human proteins such as hIL-5 have been fused with Fc portions for use ina high-throughput screening assay to identify an antagonist of hIL-5.See D. Bennett et al., Journal of Molecular Recognition Vol. 8: 52-58(1995), and K. Johanson et al., The Journal of Biological Chemistry Vol.270, No. 16, pages 9459-9471 (1995).

(1-2) Functions

The following describes detailed functions of the protein of the presentinvention, taking above-described protein SITH-1 as an example.

As shown in the later-described Example, an SITH-1 gene was expressed atall times in the cytoplasm of cells latently infected with HHV-6, butnot in productively infected cells. The gene encoding the protein SITH-1is encoded by DNA which forms a complementary strand to the previouslyreported HHV-6 latent infection specific gene (H6LT), and expression ofthe gene is enhanced at the intermediate stage of latent infection withHHV-6.

From these facts, the protein SITH-1 is considered to be a protein thatis expressed specifically during latent infection with HHV-6. Further,the protein SITH-1 has been found to be clearly different from theheretofore-identified proteins that are involved in latent infectionwith HHV-6.

Further, the present inventors proceeded functional analysis of theprotein SITH-1, and found the following fact: the protein SITH-1 bindsto CAML (calcium-modulating cyclophilin ligand, Accession #; U18242),which is a host protein, so as to increase a calcium concentration inglial cells such as astrocytes. CAML is a protein that occurs abundantlywithin a brain and lymphocytes in a host's living body, and is known toincrease a calcium concentration in cells. In addition, the increase inthe intracellular calcium concentration due to the expression of theprotein SITH-1 is considered to induce activation of general signaltransduction within the latently infected cell, thereby contributing toefficient reactivation of HHV-6.

By the term “glial cells” as used herein are meant all kinds of glialcells including mature and precursor forms of glial cells in a centralnervous system, as exemplified by astrocytes, oligodendrocytes,microglias, and ependymal cells. Other types that may be encompassed aresatellite cells, Schwann cells, and terminal gliocytes in a peripheralnervous system.

HHV-6 is known to cause latent infection of glial cells (e.g.,astrocytes) in a brain. It is believed that the calcium concentration inglial cells (e.g., astrocytes) rises, if HHV-6 being at a stage oflatent infection or an intermediate stage, which is a latent infectionstate characterized by high activity, causes SITH-1 to be expressed. Asa result of findings recently made in the mental science fields, anincrease of the intracellular calcium concentration within brain cellsis considered to be closely related to mood disorders and other mentaldisorders.

In fact, as shown in the Example, expressing the protein SITH-1 in mouseglial cells (e.g., astrocytes) turned out to induce symptoms similar tothose of mood disorders, which are mental disorders, and to increasesensitivity. This strongly suggests the possibility that HHV-6 latentlyinfecting glial cells (e.g., astrocytes) can trigger a mental disordervia the protein SITH-1.

Furthermore, HHV-6 can infect not only astrocytes but also other typesof glial cells such as microglia. Therefore, mental disorders such asdepression and manic-depressive illness may be caused by other types ofglial cells in addition to astrocytes.

The above findings show that the protein of the present invention hasability to retain activity for binding to CAML, which is a host protein,and for increasing the intracellular calcium concentration. It has alsobeen found that a mental disorder can be induced by causing the proteinof the present invention to be expressed in glial cells (e.g.,astrocytes) where the strongest expression of this protein is likely tooccur. Thus, the protein of the present invention is considered to haveability to cause a mental disorder in the host by being expressed duringlatent infection with the herpes virus or at an early stage of itsreactivation.

(1-3) Methods for Obtaining Gene and Protein

Methods for obtaining (or producing) the gene and the protein of thepresent invention are not specifically limited. The following describestypical examples of the methods.

<Method for Obtaining Protein>

As described above, the method for obtaining the protein of the presentinvention (or the method for producing the protein) is not particularlylimited. Examples of the method encompass a method for simplepurification from biological samples (e.g., cells, tissues, or anindividual organism) containing the protein of the present invention.Also, the method for purification is not particularly limited, and maybe performed in such a manner that an extract solution is extracted fromcells or tissues by a known method, and the extract solution is thenpurified by a known method (e.g., a method using a column). For example,the protein of the present invention can be purified and isolated byperforming a high performance liquid chromatography (HPLC) with respectto a crude protein fraction extracted from cells or tissues.

Further, other examples for the method for obtaining the protein of thepresent invention encompass a method using e.g., a gene recombinationtechnique. In this case, for example, the following method can beadopted: The gene of the present invention is incorporated into e.g., avector, the vector is then transferred into a host cell by a knownmethod so as to be capable of being expressed therein, and the proteinobtained by translation within the cell is purified. Specific methodsfor transfer of the gene (transformation), the expression of the gene,and the like will be described later.

Note that, for transfer of a foreign gene into a host as above, a vectorand a host may be selected depending on its purpose, since there arevarious kinds of hosts and expression vectors including a promoter whichfunctions in the host for expression of the foreign gene. The method forpurifying a produced protein differs depending on the host used and/orthe characteristics of the protein. However, use of a tag allows atarget protein to be purified in a relatively easy manner, for example.

A method for producing the mutant protein is also not limited to anyspecific one. A known mutant protein producing method can be used, forexample, site-directed mutagenesis (Hashimoto-Gotoh, Gene 152, 271-275(1995), and others), a method for producing a mutant protein byintroducing point mutation into a nucleotide sequence through the PCRtechnique, or a method for producing a mutant line by insertion oftransposon. By using any of these methods, it is possible to produce themutant protein by causing, in a nucleotide sequence of cDNA encoding theprotein (a), a mutation of a substitution, deletion, insertion, and/oraddition of one or several nucleotide. Further, the mutant protein maybe produced by using a commercially-available kit.

The method for obtaining the protein of the present invention is notlimited to the above ones. Alternatively, for example, a chemicalsynthesis using e.g., a commercially-available peptide synthesizer maybe used. Further alternatively, for example, a cell-free proteinsynthesis solution may be used for synthesizing the peptide of thepresent invention from the gene of the present invention.

<Method for Obtaining Gene>

As described above, the method for obtaining the gene of the presentinvention (or the method for producing the gene) is also notparticularly limited, and may be, for example, a method using adifferential screening (subtraction cloning). This method may beperformed in such a manner that, according to a known technique, directhybridization is repeatedly performed in a test tube so as to condensetarget cDNA (the gene of the present invention).

Each step in the differential screening may be performed underconditions conventionally applied. As for a clone obtained as a resultof this, a restriction enzyme map may be created, and a nucleotidesequence (sequencing) may be determined, for more detailed analysis ofthe clone. This analysis makes it possible to easily confirm whether ornot a DNA fragment including the sequence of the gene of the presentinvention is obtained.

Alternatively, the method for obtaining the gene of the presentinvention may be a method for isolating and cloning, according to aknown method, a DNA fragment including the gene of the presentinvention. For example, a probe which specifically hybridizes with partof the sequence of the cDNA may be prepared, and screening of a genomicDNA library or a cDNA library may be performed. The probe may have anysequence and/or length, as long as it specifically hybridizes with atleast part of the sequence of the cDNA or its complementary sequence.

Further alternatively, the method for obtaining the gene of the presentinvention may be a method using amplification means such as PCR. Forexample, primers are respectively prepared based on the 5′ and 3′ endsof a cDNA sequence (or its complementary sequence) of the gene of thepresent invention, and the primers are used to perform e.g., PCR with agenomic DNA (or cDNA) as a template, so that a DNA region between theprimers is amplified. In this way, DNA fragments including the gene ofthe present invention can be obtained in mass quantity.

Still alternatively, a polynucleotide having the sequence may besynthesized by a known chemical synthesis, based on gene sequenceinformation.

(2) Antibody of the Present Invention

The antibody of the present invention is obtained as a polyclonal ormonoclonal antibody by a known method using, as an antigen, the proteinof the present invention (e.g., the protein (a) or (b)) or a partialpeptide thereof. Examples of the known method include those that aredescribed in documents such as: Harlow et al., “Antibodies: A laboratorymanual (Cold Spring Harbor Laboratory, New York (1988); and Iwasaki etal., “Tankurohn koutai haiburidoma to ELISA (Monoclonal AntibodyHybridomas and ELISA)”, Kodansha (1991)). The antibody thus obtained maybe utilized in detecting and assaying the protein of the presentinvention.

For example, the epitope-bearing peptide of the present inventiondescribed in the above (1-1) is used to induce an antibody by a methodknown in the art. For example, see: Chow, M. et al., Proc. Natl. Acad.Sci. USA 82: 910-914; and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985). Generally, animals can be immunized with a freepeptide; however, an anti-protein antibody titer can be increased bybooster immunization by coupling of a peptide to a high-molecularcarrier (e.g., keyhole limpet hemocyanin (KLH) or tetanus toxoid). Forexample, a peptide containing cysteine can be coupled to a carrier withuse of a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS), whereas other peptides can be coupled to carriers with use ofmore general linkers such as glutaraldehyde. Animals such as rabbits,rats, and mice are immunized with a free or a carrier-coupling peptide,for example, by intraperitoneal and/or intradermic injection ofapproximately 100 μg of an emulsion including a peptide or a carrierprotein and Freund's adjuvant. Some booster immunization injections maybe required e.g., at 2-week intervals, for example, for providing ananti-protein antibody having a useful titer which is detectable in anELISA assay using a free peptide adsorbed to a surface of a solid. Ananti-protein antibody titer in the serum from an immunized animal can beincreased by selection of an anti-protein antibody, e.g., by adsorptionto a peptide on a solid support by a method known in the art and bydissolution of the selected antibody.

The term “antibody” herein means immunoglobulins (IgA, IgD, IgE, IgG,IgM and their Fab fragments, F(ab′)₂ fragments, and Fc fragments);examples of which include, but are not limited to, polyclonalantibodies, monoclonal antibodies, single-chain antibodies,anti-idiotype antibodies, and humanized antibodies.

The term “antibody that recognizes a protein of the present invention”herein is intended to encompass complete molecules and antibodyfragments (e.g., Fab and F(ab′)₂ fragments) that are capable ofspecifically binding to the above-described protein of the presentinvention. The Fab and F(ab′)₂ fragments, each of which lacks an Fcfragment included in an intact antibody, are cleared more rapidly fromcirculation, and may hardly have specific tissue binding of the intactantibody (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)). For thisreason, these fragments are preferable.

Further, another antibody capable of recognizing the protein of thepresent invention may be produced by a two-step procedure through use ofan anti-idiotype antibody. This method takes advantage of the fact thatan antibody itself is an antigen; therefore this method is capable ofgiving an antibody binding to a second antibody. According to thismethod, an antibody specific to the protein of the present invention isused to immunize animals (preferably, mice). Subsequently, spleen cellsof the animals are used to produce hybridoma cells, which are thensubjected to screening for identifying a clone producing an antibodywhose ability to bind to the antibody specific to the protein of thepresent invention can be blocked by a protein antigen of the presentinvention. Such the antibody may be an anti-idiotype antibody againstthe antibody specific to the protein of the present invention, and maybe used to immunize animals for inducing formation of further antibodiesspecific to the protein of the present invention.

It is clear that the Fab fragment, the F(ab′)₂ fragment, and otherfragments of the antibody of the present invention may be used accordingto the methods disclosed herein. These fragments are produced bycleavage caused by proteolysis using an enzyme, typical examples ofwhich encompass papain (giving an Fab fragment) or pepsin (giving anF(ab′)₂ fragment). Alternatively, a protein-binding fragment of thepresent invention can be produced by application of a recombinant DNAtechnique or through synthetic chemistry.

In detection of an increased level of the protein of the presentinvention using in vivo imaging for the purpose of diagnosis on humans,it can be preferable to use a “humanized” chimeric monoclonal antibody.Such the antibody can be generated using a genetic construct derivedfrom hybridoma cells that generate the above-mentioned monoclonalantibody. Methods for generating chimeric antibodies are known in theart of interest. For general descriptions thereof, see: Morrison,Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986);Cabilly et al. U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496;Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson etal., WO 8702671; Boulianne et al., Nature 312: 643 (1984); and Neubergeret al., Nature 314: 268 (1985).

(3) Recombinant Expression Vector of the Present Invention

The recombinant expression vector of the present invention includes thegene of the present invention encoding the protein (a) or (b). Therecombinant expression vector may be, for example, a recombinantexpression vector into which cDNA has been inserted. The recombinantexpression vector may be produced by using e.g., a plasmid, a phage, ora cosmid (not limited to these). Further, a production method of therecombinant expression vector may employ a known method.

The vector is not limited to any specific kind, and may be any one aslong as it is capable of being expressed in a host cell (host). That is,the expression vector may be one prepared as follows: In order that agene is surely expressed, a promoter sequence is selected as neededaccording to the type of the host cell; and the promoter sequence thusselected and the gene of the present invention are incorporated intoe.g., a plasmid of various kinds. Examples of the expression vectorencompass: phage vectors; plasmid vectors; virus vectors; retrovirusvectors; chromosome vectors; episome vectors; and virus-derived vectors(for example, vectors derived from bacterial plasmids, bacteriophages,yeast episomes, yeast chromosome elements, viruses (e.g., baculoviruses,papovaviruses, vaccinia viruses, adenoviruses, avipoxviruses,pseudorabies viruses, herpesviruses, lentiviruses, and retroviruses),and combinations thereof, e.g., cosmids and phagemids).

Generally, introduction of the plasmid vector is performed in sedimentssuch as calcium phosphate sediments or in a complex with charged lipids.In a case where the vector is a virus, the vector can be packaged invitro using an appropriate packaging cell line, and can subsequently betransduced into a host cell. The retrovirus vector may be replicable orreplication-defective. In the latter case, propagation of the virusgenerally occurs only in a complementary host cell.

Further, vectors each including a cis-acting regulating region for atarget gene are preferable. An appropriate trans-acting factor may besupplied by a host, by a complementary vector, or by the vector itselfduring introduction of the vector into the host. In a preferableembodiment in this regard, vectors each providing specific expressionwhich may be inducible and/or cell-type specific are preferable.Particularly preferred among such vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutritional additives.

Examples of a preferable bacteria vector to be used encompass: pQE70,pQE60, and pQE-9 (available from Qiagen); pBS vector, Phagescriptvector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A (available fromStratagene); and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (availablefrom Phrmacia). Further, examples of a preferable eukaryote vectorencompass pWLNEO, pSV2CAT, pOG44, pXT1, and pSG (available fromStratagene); and pSVK3, pBPV, pMSG, and pSVL (available from Phrmacia).

Various kinds of markers may be used to confirm whether or not the geneof the present invention has been transferred into the host cell, and toconfirm whether or not the gene is surely expressed in the host cell.That is, the expression vector preferably includes at least oneselection marker. Examples of such a selection marker encompass:dihydrofolic acid reductase or neomycin resistance for eukaryote cellculture; and drug resistance genes such as a tetracycline-resistant geneand an ampicillin-resistance gene for culture of E. coli and otherbacteria. Another example uses, as a marker, a gene deleted in a hostcell, and introduces, as an expression vector, a plasmid or the likeincluding the marker and the gene of the present invention into the hostcell. From expression of the marker gene, it is possible to confirm thatthe gene of the present invention has been transferred. Alternatively,the protein of the present invention may be expressed as a fusionprotein. For example, with Green Fluorescent Protein (GFP) derived fromAequorea victoria used as a marker, the protein of the present inventionmay be expressed as a GFP fusion protein. Further, the gene of thepresent invention may be bound to a vector including a selection markerfor propagation in the host cell.

Further, it is preferable that a DNA insert is operably linked to anappropriate promoter (e.g., phage λPL promoter, E. coli lac promoter,trp promoter, tac promoter, SV40 early promoter and late promoter, and apromoter of retrovirus LTR). As another appropriate promoter, any oneknown to a person skilled in the art may be used.

In the present invention, known bacteria promoters which are preferablyused encompass E. coli lad and lacZ promoters, T3 promoter and T7promoter, gpt promoter, λPR promoter and λPL promoter, and trp promoter.Suitable eukaryote promoters encompass CMV immediate-early promoter, HSVthymidine kinase promoter, early SV40 promoter and late SV40 promoter, apromoter of retrovirus LTR (e.g., a promoter of Rous sarcoma virus(RSV)), and metallothionein promoter (e.g., mouse metallothionein Ipromoter).

It is preferable that the recombinant expression vector furtherincludes: sites for transcription start and transcription termination;and a transcription region containing a ribosome-binding site fortranslation. A matured transcript expressed by a vector constructincludes a coding region containing (i) transcription start AUG at thestart of a polypeptide to be translated and (ii) a stop codon which isproperly positioned at the end of the polypeptide.

Transcription of DNA by a higher eukaryote may be enhanced by insertionof an enhancer sequence into a vector. The enhancer is a DNA cis-actingelement (generally, approximately 10 bp to 300 bp) which works forenhancing transcriptional activity of a promoter of a predetermined hostcell type. Examples of the enhancer encompass: SV40 enhancer (positionedat 100 bp to 270 bp on the late side of a replication origin); an earlypromoter enhancer of a cytomegalovirus; a polyoma enhancer on the lateside of a replication origin; and an adenovirus enhancer.

The above host cell is not limited to any specific one, and aconventionally-known cell of various kinds may suitably be used. Typicalexamples of an appropriate host encompass: bacterial cells (e.g., E.coli cells, Streptomyces cells, and Salmonella typhimurium cells);fungus cells (e.g., yeast cells); insect cells (e.g., Drosophila S2cells and Spodoptera Sf9 cells); animal cells (e.g., CHO cells, COScells, and Bowes melanoma cells); and plant cells. More specificexamples thereof encompass not only mammal cells such as human cells andmouse cells but also cells derived from Bombys mori, insects such asDrosophia melanogaster, bacteria such as E. coli (Escherichia coli),yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe), andCaenorhabditis elegans, and oocyte cells of Xenopus laevis. However, thepresent invention is not limited to these. A culture medium andconditions suitable for each of the above host cells may be ones knownin the art.

A method for introducing the expression vector into the host cell, i.e.,a method for transformation is also not limited to any specific one, anda conventionally-known method may suitably be used, for example,electroporation, a calcium phosphate method, a liposome method, a DEAEdextran method, microinjection, cationic lipid-mediated transfection,electroporation, transduction, or infection. These methods are describedin many standard laboratory manuals, for example, Davis et al., BasicMethods In Molecular Biology (1986).

Note that the present invention can also provide (i) a recombinantexpression vector including a polynucleotide encoding a partial fragmentof the protein of the present invention and (ii) a transformant (hostcell) genetically modified by the recombinant expression vector, each ofwhich is for recombinantly producing a partial fragment (fragment) ofthe protein of the present invention.

Further, the present invention may also encompass an invention relatedto production of the protein of the present invention or a fragmentthereof by means of the above recombinant techniques. That is, thepresent invention may also encompass a method for producing the proteinof the present invention and its fragment through use of a recombinanttechnique. A recombinant protein produced by the technique may becollected and purified from a recombinant cell culture product by meansof a known method, e.g., an ammonium sulfate precipitation or ethanolprecipitation, acid extraction, anion- or cation-exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxyapatite chromatography,or lectin chromatography. Most preferable one used for purification ishigh-performance liquid chromatography (“HPLC”).

(4) Transformant of the Present Invention

The transformant of the present invention is a transformant into whichthe gene of the present invention has been transferred, i.e., atransformant into which the recombinant expression vector described inthe above (3) has been transferred. The expression “gene has beentransferred” herein means that the gene has been transferred into atarget cell (host cell) in an expressible manner by means of a knowngene engineering method (genetic manipulation technique). Further, the“transformant” means not only a cell, a tissue, and an organ but also anindividual organism.

A method for preparing (producing) the transformant of the presentinvention may be, for example, a method for transforming the aboverecombinant expression vector. An organism to be transformed is also notlimited to any specific one, examples of which encompass various kindsof microorganisms and animals (e.g., a transgenic mouse) exemplified inthe above descriptions concerning the host cell. Further, with apromoter and/or a vector selected, a plant can also be a subject to betransformed.

(5) Gene Detection Instrument of the Present Invention

A gene detection instrument of the present invention uses, as a probe,at least part of a nucleotide sequence, or its complementary sequence,of the gene of the present invention. The gene detection instrument canbe used to e.g., detect and/or measure an expression pattern of the geneof the present invention under various conditions.

The gene detection instrument of the present invention may be, forexample, a DNA chip including a substrate (support) on which the probespecifically hybridizing the gene of the present invention isimmobilized. The “DNA chip” herein primarily refers to a synthetic DNAchip which uses, as the probe, a synthesized oligonucleotide. Not onlythat, the term “DNA chip” herein also encompasses an attachment-type DNAmicroarray which uses, as the probe, cDNA such as a PCR product.

A sequence used as the probe may be determined by a conventionally-knownmethod for determining a characteristic sequence from a cDNA sequence.Specifically, for example, the method may be a Serial Analysis of GeneExpression (SAGE) method (Science 276:1268, 1997; Cell 88:243, 1997;Science 270:484, 1995; Nature 389:300, 1997; U.S. Pat. No. 5,695,937).

Note that the DNA chip may be manufactured by a known method. Forexample, in order to use a synthesized oligonucleotide as theoligonucleotide, a photolithography technique and a solid-phase DNAsynthesis technique may be used in combination so that theoligonucleotide is yielded through synthesis on a substrate. On theother hand, in order to use cDNA as the oligonucleotide, the cDNA may beattached on a substrate with an arrayer.

Further, as well as in conventional DNA chips, a detection accuracy fora gene may be further enhanced by providing a perfect-match probe(oligonucleotide) together with a mismatch probe, which differs from theperfect-match probe by a single base substitution. Further, in order todetect different genes in parallel, a DNA chip may be configured suchthat a plurality of kinds of oligonucleotides are immobilized on asingle substrate.

The following describes the gene detection instrument of the presentinvention in greater detail.

<Substrate>

A material of the substrate for use in the gene detection instrument ofthe present invention only needs to be one on which an oligonucleotidecan stably be immobilized. Examples of the material encompass, but arenot limited to, synthetic resins (e.g., polycarbonate and plastic) andglass. A shape of the substrate is also not limited to any specific one.For example, a plate-shaped substrate or a film-shaped substrate maypreferably be used.

<Oligonucleotide to be Immobilized on Surface of Substrate>

The oligonucleotide to be immobilized on the surface of the substrate ofthe gene detection instrument of the present invention only needs to bean oligonucleotide which is based on at least part of the nucleotidesequence of the gene of the present invention. By establishment ofhybridization between the oligonucleotide and a nucleic acid derivedfrom a sample, it is possible to detect a gene contained in the sample.Note that the oligonucleotide which is based on at least part of thenucleotide sequence of the gene of the present invention will behereinafter referred to as “capture oligo” in some cases.

The capture oligo may be designed based on the nucleotide sequence ofthe gene of the present invention. Thus, the capture oligo may be thenucleotide sequence itself, or may include a mutation as long as itallows establishment of specific hybridization between the capture oligoand a nucleic acid prepared from a sample to be detected. The positionof the mutation is not particularly limited.

A length (the number of bases) of the capture oligo is not particularlylimited. However, if the length is too short, detection of thehybridization becomes difficult; if the length is too long, non-specifichybridization is allowed. The present inventors kept studyingoptimization of the length of the capture oligo, and determined 12 to 50base length as a standard length. The standard length is preferably 12to 40 base length, more preferably 12 to 30 base length, furtherpreferably 13 to 22 base length. However, the present invention is notlimited to this. The base length depends primarily on sequencecharacteristics (a content of a certain base, repetition of a certainbase). Further, the present inventors have confirmed that even ashort-chained capture oligo is capable of specific hybridizationprovided that the short-chained capture oligo has a fine binding.

In a case where the capture oligo has any of a hair-pin structure, aloop structure, and other tertiary structures each of which hindershybridization with a nucleic acid derived from a sample, substitutingone or more nucleotides constituting the capture oligo with inosine or anucleic acid(s) not paired with any nucleotide(s) can cancel thetertiary structure.

A synthesis method of the capture oligo is not limited to any specificone. For example, a known method (e.g., the method described inManiatis, T. et al., Molecular Cloning A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1989)) may be used.Generally, the capture oligo can be chemically synthesized with use of acommercially-available DNA synthesizer.

In the gene detection instrument of the present invention, it ispreferable that a so-called control capture oligo as well as theoligonucleotide which is based on at least part of the nucleotidesequence of the gene of the present invention are immobilized on thesurface of the substrate. The control capture oligo includes a positivecontrol capture oligo and a negative control capture oligo. The positivecontrol capture oligo is used to check whether or not an amplificationreaction is successfully proceeding in the later-described probepreparing step. The negative control capture oligo is used to check fornon-specific hybridization, i.e., a false-positive hybridization signal.The present invention also encompass a gene detection instrument inwhich these positive control capture oligo and negative control captureoligo are immobilized on the surface of the substrate.

The positive control capture oligo may be any oligonucleotide, as longas it is designed based on a nucleotide sequence included in a probeprepared from a sample to be detected. Further, in order that aplurality of samples to be detected are detected at once with use of asingle gene detection instrument, positive control capture oligos may bedesigned respectively for the samples to be detected, or a positivecontrol capture oligo may be designed based on a nucleotide sequenceshared by probes prepared from the plurality of samples to be detected.In case there is no nucleotide sequence shared by the probes preparedfrom all of the samples to be detected, a positive control capture oligomay be designed for each of some groups. Alternatively, an artificialsequence may be designed so that it has a different sequence from asequence of a subject bacterium but has a common primer sequence, and apart of the artificial sequence may be used as a positive controlcapture oligo. With such an artificial sequence used as a template, aprobe can be prepared (such a probe is herein called “control probe”),and the resulting probe is added to a probe prepared from a sample. Inthis way, specificity of the hybridization can be tested. More detailson the probe will be discussed below.

It is preferable that the negative control capture oligo is designedsuch that it has a nucleotide sequence of a positive control captureoligo with an artificial substitution of one or more bases but less than20% of the total bases of the sequence. The number of substituted basesis determined taking into consideration of hybridization conditions sothat the negative control capture oligo does not hybridize with theprobe derived from the sample to be detected.

The sample to be detected is not limited to any specific one. Further,the number of kinds of capture oligos to be immobilized on one substrateonly needs to be one or more, and there is no upper limit for it. Also,it is most preferable that the gene detection instrument of the presentinvention is designed to be a so-called microarray type in which aplurality of partial fragments (having different nucleotide sequences)of the gene of the present invention are immobilized on one substrate ascapture oligos.

<Immobilization of Oligonucleotide (Capture Oligo)>

A method for immobilizing the oligonucleotide on the surface of thesubstrate is not limited to any specific one, but may be selected fromknown methods as needed. For example, means used for generalhybridization methods, e.g., physical adsorption, electrical bonding, ormolecular covalent bonding, is available. For the gene detectioninstrument of the present invention, it is preferable to use a substratehaving a carbodiimide group or an isocyanate group on its surface (U.S.Pat. No. 5,908,746, Tokukaihei, No. 8-23975) for immobilization.

If an amount of the oligonucleotide spotted on the substrate is toosmall, detection may be difficult because there may not be enoughreaction between the oligonucleotide and the probe. Further,high-integration spotting brings about technical problems and a highcost, and also requires an expensive higher-precision detectioninstrument (e.g., a scanner) for detecting a hybridization signal byusing e.g., a fluorescent label of the probe or chemiluminescence.Therefore, it is preferable to immobilize, on the surface of thesubstrate, the oligonucleotide within a size of 10 μm to 1,000 μm indiameter. A method for spotting the oligonucleotide onto the substrateis not limited to any specific one. For example, spotting can beperformed by spotting a solution of the oligonucleotide onto thesubstrate using a spotting machine. In this way, the oligonucleotidesolution can be generally spotted substantially in a circle.

(6) Detection Instrument Using Protein of the Present Invention or itsPartial Fragment

A detection instrument of the present invention uses, as a probe, atleast part of the amino acid sequence of the protein of the presentinvention. In other words, the detection instrument of the presentinvention is a detection instrument having the protein of the presentinvention or its partial fragment (fragment) immobilized thereto. Thedetection instrument can be used to detect and/or measure, under variousconditions, a substance (e.g., a polypeptide, a nucleic acid, or anantibody) which interacts with the protein of the present invention.

The detection instrument of the present invention may be, for example,one including a substrate (support) having the probe immobilizedthereto, which probe specifically binds to an antibody recognizing theprotein of the present invention. For an amino acid sequence used as theprobe, it is preferable to use a site of the protein of the presentinvention which site specifically interacts with the antibody of thepresent invention, i.e., an epitope-bearing peptide of the protein ofthe present invention.

A material of the substrate for use in the detection instrument of thepresent invention only needs to be one on which an oligopeptide canstably be immobilized. Examples of the material encompass, but are notlimited to, synthetic resins (e.g., polycarbonate and plastic) andglass. A shape of the substrate is also not limited to any specific one.For example, a plate-shaped substrate or a film-shaped substrate maypreferably be used.

Further, a method for immobilizing an oligopeptide on the substrate maybe a conventionally-known method, and is not limited to any specificone. For example, immobilization may be performed by: a method in whichan oligopeptide is bound to an insoluble carrier by means of a covalentbonding method or an adsorption method; an entrapping immobilizationmethod in which an oligopeptide is surrounded by high-molecularsubstances; or a method in which an oligopeptide is immobilized on asupport by using a cross-linking agent or the like. Note that a suitableimmobilizing method may be selected considering (i) compatibilitybetween a substrate for immobilization and an oligopeptide and (ii) apurpose of use of an immobilized substance.

(7) Usefulness of Gene, Protein, and Others of the Present Invention

As described above, the gene and the protein of the present inventionare expressed specifically during latent infection with a herpesvirus.This protein is considered to have ability to induce a mental disorderin a host by being expressed in glial cells (e.g., astrocytes) in thebrain.

Further, interestingly, the gene and the protein of the presentinvention have been found to be related to patients with mentaldisorders. Stated in greater detail, as shown in the later-describedExample, an anti-SITH-1 antibody was detected in about 50% of patientssuffering from mood disorders or other mental disorders, whereas theanti-SITH-1 antibody was hardly detectable in healthy persons (thefrequency of detection of the anti-SITH-1 antibody in healthy personswas less than about 2%).

Thus, the present inventors have discovered on their own that anantibody specific to the protein of the present invention is foundsignificantly only in patients with mood disorders and other mentaldisorders, but is hardly detectable in healthy persons. Note that the“mental disorder” as used herein is intended to mean such a state thatthe daily life or social life undergoes considerable limit on account ofdisorders in mental functions such as consciousness, intelligence,memory, emotions, thought, and behavior. The “mood disorder” is intendedto mean such a state that because of persistent mood or emotionalchanges, abnormally depressive or related feelings are experienced tobring disturbances into daily life functioning or social lifefunctioning.

Specific reasons for the above state where “an antibody specific to theprotein of the present invention is found significantly only in patientswith mood disorders and other mental disorders, but is hardly detectablein healthy persons” are now being under a diligent study to beunraveled. The protein of the present invention has such a nature thatit is actively produced at the intermediate stage where latent infectionis induced toward reactivation. It is believed that in response to astress, reactivation of herpes viruses (e.g., HHV-6) is induced, wherebythe protein of the present invention is produced. Persons who have theantibody against the protein of the present invention, accounting forabout 50% of patients with mental disorders, are considered to have thatprotein expressed abundantly due to stress or any genetic factor for aprolonged period in glial cells (e.g., astrocytes) in the brain, whichglial cells are latently infected with HHV-6. As a result, an increaseof a calcium concentration in glial cells (e.g., astrocytes) continuesfor a prolonged period, and serotonin metabolism and other importantfunctions of glial cells (e.g., astrocytes) are impaired, whereby amental disorder would manifest itself. Chronic fatigue syndrome (CFS)patients who present with mental disorders carry the antibody againstthe protein of the present invention with a high frequency. The reasonfor this would be that the CFS patients are often latently infected withgreater numbers of HHV-6 than healthy persons are, thus having a greaterlikelihood for the production of the protein of the present invention(protein SITH-1). The fact that the CFS patients are latently infectedwith greater numbers of HHV-6 than healthy persons are is also supportedby the result of the reaction between the previously reported latentinfection specific gene product and the antibody in CFS patients (seeNon-Patent Document 5).

Thus, although a detailed mechanism of the phenomenon that the antibodyagainst the protein of the present invention is found in patients withmental disorders has not been unraveled yet, utilizing this phenomenonprovides a determination method and a diagnosis method contributing toobjective diagnosis of mental disorders. Further, the present inventionalso relates to a determination kit, a diagnosis kit, an animal modelproducing method, and a drug screening method. The following describeseach of these methods in detail.

(7-1) Determination Method of the Present Invention

The determination method of the present invention only needs to be amethod for determining whether or not an antibody recognizing theprotein of the present invention (i.e., the protein (a) or (b)) existsin a subject. Note that the term “subject” means a human or a mammalother than a human.

An assay for an antibody is enabled by detection through, for example, areaction for binding to a protein recognized by the antibody or apartial fragment of the protein. Thus, in this determination method, aprotein recognized by the antibody or a partial fragment of the proteinis preferably used to determine for the presence of the antibody in animmunological manner (i.e., using an antigen-antibody reaction). Notethat the “partial fragment” preferably contains at least anepitope-bearing peptide.

To give an example of this determination method, an insoluble carrier onwhich the protein according to the present invention or a partialfragment thereof is immobilized is brought into contact with abiological sample taken from a subject and washed, and then antibodiesspecifically bound to the protein or the partial fragment thereof on theinsoluble carrier are detected. The antibodies specifically bound to theprotein or the partial fragment thereof on the insoluble carrier are,for example, antibodies derived from the subject. Therefore, such theantibodies can be easily detected using a secondary antibody, i.e., anantibody specific to the antibodies in the subject. In this case, a dye,an enzyme or a radioactive or fluorescent label may be incorporated inthe secondary antibody so as to enhance and thereby further facilitatethe intended detection.

Thus, antibody assays to be used in the determination method underconsideration include assay techniques that make use of traditionalimmunohistological approaches such as a fluorescent antibody technique,a dot blot assay, a western blotting technique, enzyme-linkedimmunosorbent assay techniques (including ELISA and a sandwich ELISAtechnique), a radioimmunoassay technique (RIA), and an immunodiffusionassay technique. These assays use molecules such as avidin and biotinfor the purposes of molecular immobilization and detection, andtechniques for preparing these reagents and methods of use thereof maybe technologies known to a person skilled in the art. Note that theresult of the determination method under consideration is animmunohistological stain of tissue sections for pathological testing.

Note also that the determination method under consideration ispreferably performed using a biological sample isolated from thesubject. The term “biological sample isolated” may cover any sample thatcontains cells, tissues or disrupted pieces thereof as taken from thesubject. For example, the “biological sample isolated” may be any ofperipheral blood, saliva, urine, stools, and cell samples, and is notlimited to any specific one. Among these, particularly preferable sampleis peripheral blood taken from the subject, in view of the fact thatherpes viruses latently infect macrophages in peripheral blood. In thiscase, the subject benefits from a low degree of invasion.

An amount of antibodies present in a biological sample (sample) can bereadily calculated by making comparison with an amount of antibodiespresent in a standard preparation (e.g., a standard sample taken from ahealthy person or one taken from a typical patient with a mentaldisorder), using e.g., a linear regression computer algorithm. Whilevarious assay techniques are available for antibody detection, anexample for ELISA is described in Iacobelli et al., Breast CancerResearch and Treatment 11: 19-30 (1988).

Suitable enzyme labels may be exemplified by those derived from a classof oxidases which catalyze the generation of hydrogen peroxide throughreaction with the substrate. Glucose oxidase is particularly preferable,since it has satisfactory stability and its substrate (glucose) iseasily available. Activity of the oxidase label can be assayed bymeasuring a concentration of hydrogen peroxide formed by anenzyme-labeled antibody/substrate reaction. In addition to enzymes,other suitable labels include radioisotopes (e.g., iodine (¹²⁵I and¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), andtechnetium (^(99m)Tc), as well as fluorescent labels (e.g., fluoresceinand rhodamin) and biotin.

A level of antibodies (against the protein of the present invention)that are present in biological samples obtained from the subject canalso be detected in vivo by methods other than the above-describedimmunoassay technique, for example, by image analysis. In short, in viewof the fact that the antibody against the protein according to thepresent invention is also a protein, an antibody that specificallyrecognizes this antibody may be used for in vivo detection by imageanalysis of the level of the antibodies (against the protein of thepresent invention) that are present in the biological samples obtainedfrom the subject.

Antibody labels or markers for the in vivo image analysis of antibodiesencompass those that can be detected by X-ray imaging, NMR, or ESR. ForX-ray imaging, suitable labels encompass radioisotopes such as barium orcesium that emit detectable radiation but that are clearly harmless tothe sample under test. Suitable markers for NMR and ESR encompass thosewhich can be used to label a nutrient for culturing an associatedhybridoma to produce a corresponding antibody, whereby the label isincorporated in the antibody produced; an example of such label isdeuterium having a detectable characteristic spin.

An antibody or a fragment thereof that is specific for the antibodyagainst the protein of the present invention and that is labeled with asuitable, detectable image-analysis portion, such as a radioisotope(e.g. ¹¹¹In or ^(99m)Tc), a radio-opaque substrate or a substancedetectable by nuclear magnetic resonance is introduced (e.g.,parenterally, subcutaneously, or intravenously) into a mammal to betested for a disorder. It will be understood in the art of interest thata quantity of the image-analysis portion required for generating adiagnostic image is determined by the size of the sample under test andthe image analysis system to be used. In the case where that portion ispart of a radioisotope, a quantity of radioactivity to be injected intoa human sample is typically in a range from about 5 to about 20 mCi of^(99m)Tc. Subsequently, the label antibody or the fragment thereof isaccumulated preferentially at a site of the cell which site includingthe antibody against the protein of the present invention. Note that anin vivo image analysis of tumors is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Turner Imaging, Chapter 13: The Radiochemical Detection of Cancer,Burchiel, S. W. and Rhodes, B. A. eds., Masson Publishing Inc. (1982)).

The following lists specific examples of a label available for thepresent invention. Examples of suitable enzyme labels include malatedehydrogenase, Staphylococcus nuclease, yeast alcohol dehydrogenase,α-glycerol phosphate dehydrogenase, triose phosphate isomerase,peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotope labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S_(,) ¹⁴C_(,) ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu,²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, and ¹⁰⁹Pd. Indium 111 (¹¹¹In) is a preferredisotope in the case where in vivo imaging is employed, since this avoidsa problem of dehalogenation of a monoclonal antibody labeled with ¹²⁵Ior ¹³¹I, which dehalogenation is caused by a liver. Further, thisradionuclide has a favorable gamma release energy for imaging (Perkinset al., Eur. J. Nucl. Med. 10: 296-301 (1985); Carasquillo et al., J.Nucl. Med. 28: 281-287 (1987)). For example, indium 111 (¹¹¹In) coupledto a monoclonal antibody using 1-(P-benzyl isothiocyanate)-DPTA hasshown little uptake in non-tumorous tissues, particularly a liver, andtherefore enhances specificity of tumor localization (Esteban et al., J.Nucl. Med. 28: 861-870 (1987)).

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include ¹⁵²Eu label, fluoresceinlabel, isothiocyanate label, rhodamin label, phycoerythrin label,phycocyanin label, allophycocyanin label, o-phthalaldehyde label, andfluorescamine label.

Examples of suitable marker toxins include diphtheria toxin, ricin, andcholera toxin.

Examples of chemiluminescent labels include luminal label, isoluminallabel, aromatic acridinium ester label, imidazole label, acridinium saltlabel, oxalate ester label, luciferin label, luciferase label, andaequorin label.

Examples of contrast agents for nuclear magnetic resonance include heavymetal atomic nuclei such as Gd, Mn, and Fe.

Representative techniques for binding the above-mentioned labels toantibodies are provided by Kennedy et al. (Clin. Chim. Acta 70:1-31(1976)) and Schurs et al. (Clin. Chim. Acta 81: 1-40 (1977)). Couplingtechniques described in the latter include a glutaraldehyde method, aperiodate method, a dimaleimide method, and am-maleimidebenzyl-N-hydroxy-succinimide ester method. All of thesemethods are incorporated herein by reference.

(7-2) Diagnosis Method

The diagnosis method of the present invention only needs to use theabove determination method. Specific configuration, conditions andothers of the diagnosis method are not particularly limited. Forexample, by using as a marker the antibody of the present inventionexisting in a human subject or an animal subject, it is possible todetermine that the human subject or the animal subject contacts a mentaldisorder. Further, diagnosis may be performed by using as an indicationa quantitative value of the antibody of the present invention asfollows: A threshold value is appropriately set according to aquantitative value (normal value) measured in a healthy person or aquantitative value (disorder value) measured in a typical patient withmental disorders; if a value measured in a subject is above or below thethreshold value, the subject is determined to contract a mental disorderwith a high probability. Once a mental disorder is developed, an amountof the antibody is increased. In view of this, in the present invention,for example, a quantitative value (normal value) measured in a healthyperson is set as a threshold value; if a value measured in a humansubject is below the threshold value, the human subject can bedetermined to contract a mental disorder with a high possibility.

Note that the term “human subject” herein means a human, and the term“animal subject” means an animal other than a human. Examples of theanimal subject encompass mice, rats, and monkeys. Not only that, anyanimal other than a human can be the “animal subject”.

Thus, this diagnosis method makes it possible to easily and accuratelydetermine (i) whether or not a subject has a mental disorder or (ii)whether or not a subject has a possibility of contracting a mentaldisorder. Further, the diagnosis method for an animal subject will bequite useful for e.g., drug screening for development of therapeuticagents for mental disorders and animal subjects for testing drugeffectiveness.

(7-3) Determination Kit, Diagnosis Kit

Each of a determination kit and a diagnosis kit of the present inventiononly needs to be designed to allow the determination method described inthe above (7-1) or the diagnosis method described in the above (7-2) tobe performed. Specific configurations, materials, instruments and othersof these are not specifically limited. To be specific, in order toimmunologically detect the antibody of the present invention, each ofthe determination kid and the diagnosis kit preferably includes any of:(i) a protein of the present invention; (ii) a partial fragment(preferably including an epitope-bearing peptide) of the protein (i);and (iii) a detection instrument to which the protein (i) or the partialfragment (ii) is immobilized.

A kit having the above configuration is quite useful, since such a kitmakes it possible to easily and reliably perform the determinationmethod or the diagnosis method of the present invention.

Further, in addition to the above configuration, each of thedetermination kit and the diagnosis kit may include an item forperforming each step of the determination method or the diagnosismethod. Examples of such an item encompass: instruments for taking asample from a subject (e.g., a syringe (injector) for collectingperipheral blood); and items required for performing the determinationmethod and/or the diagnosis method such as laboratory instruments andvarious reagents (e.g., reagents used for an immunological reaction suchas ELISA). Further, each of the determination kit and the diagnosis kitmay include an arithmetic unit (e.g., a computer) or software each ofwhich is required for performing the determination more easily andaccurately.

(7-4) Methods for Producing, Determining, Screening, and EvaluatingAnimal Model

The diagnosis method of the present invention is applicable to: a methodfor producing an animal model (other than a human) of a mental disorder;a method for determining usefulness of the animal model; and a methodfor determining usefulness of a drug by means of drug screening usingthe animal model. Specifically, as described in the Example, the animalmodel of the mental disorder can be produced by introducing the proteinSITH-1 into the brain of an animal using e.g., a vector. Further,usefulness of the animal model of the mental disorder can be determinedas follows: Similarly to the determination method and the diagnosismethod, it is determined whether or not an animal subject develops amental disorder, depending on the presence or absence of the antibody ofthe present invention; if the animal subject has developed the mentaldisorder, the animal subject can be determined to be useful as theanimal model of the mental disorder.

It is more preferable that each of the above various methodsadditionally uses, as evaluation means, a diagnosis method utilizinge.g., a heretofore known behavior disorder and/or startle response of ananimal. Specifically, for diagnosis in animal tests, any of thefollowings may be employed: (i) a test for a behavior disorder, e.g., atail suspension test or a forced swimming test; and (ii) a known brainfunction test, e.g., startle response.

The “subject animal” herein may be any animal other than a human,particularly preferable examples of which encompass mice, rats, guineapigs, dogs, rabbits, monkeys, and jockos. Determination (diagnosis) ofmental disorders for animals other than a human was more difficult. Interms of this, the method of the present invention is quite useful.Further, a candidate substance for a psychotropic agent or anantipsychotic agent (an agent for treating or improving a mentaldisorder) may be administered to such an animal model, and thereafter atest for the behavior disorder and detection of the antibody of thepresent invention may be performed in a manner as described above. Then,if the mental disorder is cured or improved, the candidate substance canbe determined to have an anti-mental disorder effect. Thus, use of thediagnosis method of the present invention makes it possible to easilyand reliably perform screening for a candidate substance for apsychotropic agent. The “candidate substance for a psychotropic agent”herein may be any substance desired by a person who conducts the test.

Note that the point of the determination method, the diagnosis method orthe like of the present invention is to provide an objectivedetermination method for determining whether or not a subject contractsa metal disorder by detection of an antibody against a protein which isexpressed specifically during latent infection with a herpesvirus, anddoes not lie in each manipulation specifically described herein.Therefore, it should be noted that determination methods and diagnosismethods using manipulations other than those described above are alsoencompassed in the scope of the present invention.

In addition, infection with herpesviruses is considered to be relatedto: diseases accompanied by immunodeficiency such as CFS, which isdescribed also in the Example, (e.g., autoimmune diseases such asCrohn's disease); cutaneous diseases which are considered to beassociated with HHV-6 (e.g., drug-induced hypersensitivity syndrome);and encephalitis and encephalopathy induced by HHV-6. Therefore, itwould be considered that the determination method and the diagnosismethod of the present invention also enable objective diagnosis andevaluation of these diseases.

That is, the protein, the gene and others of the present invention canbe used as a disease marker for various diseases which might be involvedwith HHV-6.

Further, the present invention also encompasses an animal model producedby transfer of the above-described gene of the present invention, a geneproduct thereof (e.g., a protein encoded by the gene), or a recombinantexpression vector having the gene. Since the gene of the presentinvention is involved in a mental disorder as described above, theanimal model produced by transfer of the gene, the gene product thereof(e.g., the protein encoded by the gene), or the recombinant expressionvector having the gene manifests a symptom of the mental disorder.Examples of the symptom of the mental disorder encompassmanic-depressive-like symptoms, mania-like symptoms, depression-likesymptoms, and, depending on the test method, schizophrenia-likesymptoms.

A subject animal is not limited to any specific one, as long as it isavailable as a test animal. Particularly preferable one is a mammal, forexample, a mouse, a rat, or a monkey.

Furthermore, a method for transfer of the gene, the gene product, andthe recombinant expression vector may be a conventionally-known method,and is not limited to any specific one. For example, a method forcausing the protein of the present invention to be expressed in a braincan be a method using an adenovirus vector or a method using aretrovirus vector (see the later-described Example), and, of course, canbe any of methods using vectors which are not the adenovirus vector orthe retrovirus vector. Alternatively, gene transfer using a generaltransgene (e.g., production of a transgenic mouse) can be used. Furtheralternatively, a method for directly inoculating the protein of thepresent invention into a brain can be used.

The animal model of the mental disorder can be suitably used for e.g.,study on treating methods for mental disorders, study on effects ofdrugs, determination of effects of drugs, and evaluation of treatingmethods (e.g., thermotherapy) which are not treating methods usingdrugs, and therefore is quite useful.

Further, the animal model can be used for study on a factor related to adevelopment factor of a mental disorder. The animal model can also beused for research of prevention of development of mental disorders e.g.,by studying how much fatigue and stress are involved in induction of amental disorder.

The following shows Examples to describe the embodiments of the presentinvention in greater detail. Needless to say, the present invention isnot limited to the Examples below, and various forms may be taken forthe details. Further, the present invention is not limited to thedescription of the embodiments above, but may be altered by a skilledperson within the scope of the claims. An embodiment based on a propercombination of technical means disclosed in different embodiments isencompassed in the technical scope of the present invention.

Examples 1. Identification of Gene Product (mRNA) Encoding LatentInfection Specific Protein SITH-1

Messenger RNA (mRNA) was separated from those macrophages described inNon-Patent Document 1 that were latently infected with HHV-6, and areverse transcription reaction was performed using random primers, IE4RBas a primer for reverse transcription of sense transcripts, and IE2FB asa primer for reverse transcription of anti-sense transcripts.Thereafter, the resultant reverse transcripts (cDNA) were amplified bythe PCR technique using the primers IE4RB and IE2FB, and the productswere further amplified by the double-nested PCR technique using theinner primers IE4RA and IE2FA. FIG. 1 shows (i) the correspondencebetween the sense transcript (H6LT) of the known mRNA during productiveinfection and the novel latent infection specific gene and (ii) an openreading frame of the latent infection specific protein SITH-1. Fordetails of the sequence information about SITH-1 and the novel latentinfection specific gene, see the SEQUENCE LISTING.

As a result, the amplification yielded a 925-bp product, which differedboth from (i) a 351-bp product amplified from mRNA being expressed inMT-4 cells that were productively infected with HHV-6 and (ii) a 351-bpproduct amplified from a latent infection specific gene product (HHV-6latency-associated transcript: H6LT), described in Non-Patent Document3, that was detectable during latent infection of macrophages (MΦ) withHHV-6.

This product was also different from a 1241-bp product amplified fromHHV-6 DNA in that it was solely amplified from the product of thereverse transcription of the anti-sense transcripts in the cellslatently infected with HHV-6. From this, this product was shown to be aheretofore unknown, novel latent infection specific gene product (seeFIG. 2). In FIG. 2, “R” signifies a random primer, “S” signifies a sensetranscript, and “anti-S” signifies an anti-sense transcript.

To determine the structure of this novel latent infection specific genemRNA, a 5′-rapid amplification of cDNA ends (RACE) method and a 3′-RACEmethod were performed, whereby not only the 5′- and 3′-ends but also theoverall nucleotide sequence was determined (see FIG. 3).

(SEQ ID NO: 4) IE4RB: 5′-GATGCTCCTTCTTCCACATTACTGG-3′ (SEQ ID NO: 5)IE2FB: 5′-CATCCCATCAATTATTGGATTGCTGG-3′ (SEQ ID NO: 6)IE2FA: 5′-GAAACCAC-CACCTGGAATCAATCTCC-3′ (SEQ ID NO: 7)IE4RA: 5′-GACACATTCTTGGAAGCGATGTCG-3′ (SEQ ID NO: 8)N1: 5′-GCTGGGTAGTCCCCACCTTTCTAGA-3′ (SEQ ID NO: 9)αF1: 5′-CTGAAGCATGTAAGCACATCTCTTGC 3′ (SEQ ID NO: 10)αR1: 5′-GCTTCGAGATCAGTAGTGGTACG-3′

2. Functional Analysis of Novel Latent Infection Specific Gene ProteinSITH-1

To study the function of the protein SITH-1, a host protein to which theprotein SITH-1 would bind within cells was identified. This wasperformed by screening of a human fetal brain cDNA library by means ofyeast two-hybrid assay with the protein SITH-1 used as a bait. Theresult is shown in FIG. 4. In FIG. 4, (A) shows yeast clones in whichβ-galactosidase was strongly expressed owing to binding between SITH-1and CAML; (B) shows a diagram verifying by western blotting and stainingof anti-CAML antibodies that, in the in vitro pull-down assay, CAMLexpressed in E. coli could be co-precipitated with a GST-SITH-1 fusionprotein which was also expressed in E. coli; and (C) shows a diagramverifying by western blotting and staining of anti-FLAG antibodies that,after SITH-1 with a FLAG tag and CAML were transferred into 293T cellsusing an expression vector, the SITH-1 could be co-precipitated withanti-CAML antibodies. As FIG. 4 shows, the protein SITH-1 was found tobind strongly to the calcium-signal modulating cyclophilin ligand(CAML).

CAML is a protein that has been reported to show strong expression inlymphocytes and in a brain, and it is known that CAML has ability toincrease an intracellular calcium concentration. Thus, in order to seewhether the protein SITH-1 would be mediated by CAML to increase theintracellular calcium concentration, both an astrocyte-like glial cellline (U373) in which SITH-1 had been expressed and untreated U373 cellswere stained by the fluorescent antibody technique using anti-SITH-1antibodies and anti-CAML antibodies. As it turned out, when the proteinSITH-1 was expressed in the astrocyte-like glial cell line (U373), moreCAML was found than in the untreated U373 cells (FIG. 5). A level ofCAML expression in U373 cells was not very high when the U373 cells wereuntreated, but more CAML was found to occur by expressing SITH-1 in theU373 cells.

In another experiment, two samples were prepared; one sample wasprepared by transferring SITH-1 into an astrocyte-like glial cell line(U373) via a retrovirus vector, and the other sample was prepared byintroducing only the vector into U373. Each sample was stimulated withthapsigargin (TG), and an intracellular calcium concentration wasmeasured using Fura2. As a result, the actual measurement of theintracellular calcium concentration showed that on account of thestimulation with thapsigargin (TG), the intracellular calciumconcentration in the SITH-1 expressing astrocyte-like glial cell linewas considerably higher than in the cells into which only the vector hadbeen transferred (FIG. 6).

From those results, it was found that the protein SITH-1 has ability toincrease an intracellular calcium concentration in an astrocyte-likeglial cell line by being expressed during latent infection with HHV-6 toincrease an amount of intracellular CAML.

3. Relationship Between SITH-1 and Mood Disorders

In the next step, the present inventors studied the relationship betweenSITH-1 and mental disorders. The results are shown in FIG. 7. Unlike thelatent infection specific gene protein reported in e.g., Non-PatentDocument 5, the relationship between an antibody against SITH-1 andpatients with chronic fatigue syndrome was low, but the frequency ofantibody carriers was high among patients with chronic fatigue syndromeaccompanied by mental disorders. In many cases, the patients withchronic fatigue syndrome (CFS) accompanied by psychiatric symptomsprimarily manifested depressive symptoms, whereas infantile CFS patientsmainly presented with abnormal agitation. In FIG. 7, “bipolar I” refersto patients with manic-depressive illness of severe symptoms. Healthyadults scarcely carried the antibody against SITH-1. For antibody titermeasurement, SITH-1-expressing 293T cells were used as antigens, and thefluorescent antibody technique was applied.

4. Construction of Model Mice of Mental Disorder by Expressing SITH-1

SITH-1 having a glial fibrillary acidic protein (GFAP) promoter linkedupstream of its open reading frame was injected into the brains ofnewborn mice using an adenovirus vector or a retrovirus vector. GFAP isa protein expressed specifically in glial cells such as astrocytes.About four or five weeks after the injection, behavior of each mouse wasobserved to confirm that model mice with mental disorders wereestablished by transfer of SITH-1.

A tail suspension test and a forced swimming test were conducted toevaluate mental disorders; these tests are commonly used to observepatients with depression or manic-depressive illness. Specifically, miceinto which SITH-1 was transferred using an adenovirus vector weresubjected to a tail suspension test. As it turned out, the mice intowhich SITH-1 was transferred had a markedly shorter immobile time,indicating that these mice were in a manic state (FIG. 8). Subsequently,mice into which SITH-1 was transferred using a retrovirus vector weresubjected to a forced swimming test. As it turned out, the mice intowhich SITH-1 was transferred using the retrovirus vector at HIGH titerhad a longer immobile time than control mice into which an enhancedgreen fluorescence protein (EGFP) gene was transferred, indicating thatthe mice into which SITH-1 was transferred at HIGH titer were in a stateof depression. In contrast, mice into which SITH-1 was transferred usingthe retrovirus vector at LOW titer had a shorter immobile time,indicating that the mice into which SITH-1 was transferred at LOW titerwere in a manic state (FIG. 9). Thus, the manic state was observed inthe tail suspension test, whereas both the manic state and the depressedstate were observed in the forced swimming test. In addition, the factthat either the manic state or the depressed state was observeddepending on the titer of the retrovirus vector used to introduce SITH-1not only shows that the model of interest can serve as models ofdepression and manic-depressive illness alike, but also suggests that anamount of expression of SITH-1 affects symptoms of mental disorders.

The present inventors also measured a prepulse inhibition in order tocheck for any abnormality in a startle response, which abnormality is tobe found in patients with manic-depressive illness and schizophrenia.Specifically, mice into which SITH-1 was transferred using theadenovirus vector were evaluated for a startle response by measuring theprepulse inhibition. The result is shown in FIG. 10; as it turned out,the SITH-1 transferred mice had a markedly lower prepulse inhibition,indicating that these mice had become overly sensitive to stimuli. Thus,considerable abnormality was also observed in the startle response,indicating that SITH-1 greatly affects a brain function associated withmental disorders.

5. Construction 2 of Model Mice of Mental Disorder by Expressing SITH-1

Next, an open reading frame of SITH-1 was linked downstream of a GFAPpromoter, and expressed in glial cells of mice using an adenovirusvector; three weeks later, the mice were measured for their motoractivity in terms of wheel running activity. The result is shown in FIG.11.

As FIG. 11 shows, compared to control mice in which EGFP (enhanced greenfluorescence protein) was expressed, the SITH-1 expressing mice hadtheir motor activity enhanced, and the SITH-1 expressing mice showed atendency to be in a manic state.

6. Construction 3 of Model Mice of Mental Disorder by Expressing SITH-1

Subsequently, an open reading frame of SITH-1 was linked downstream of aGFAP promoter, and expressed in glial cells of mice using a lentivirusvector; eight weeks later, the mice were measured for their motoractivity in terms wheel running activity. The result is shown in FIG.12.

As FIG. 12 shows, compared to control mice in which EGFP (enhanced greenfluorescence protein) was expressed, the SITH-1 expressing mice hadtheir motor activity suppressed, and the SITH-1 expressing mice showed atendency to be in a depressed state.

As can be seen from FIGS. 11 and 12, the same SITH-1 was found to causetwo opposite phenomena, a manic state and a depressed state. The reasonswould be as follows: 1) SITH-1 carried by the adenovirus vector wasexpressed in a greater amount than when SITH-1 was carried by thelentivirus vector; 2) on the other hand, the lentivirus vector allowedSITH-1 to be expressed for a longer period, so the effect of theprolonged expression of SITH-1 was observed.

This fact, i.e., model mice of a manic state and a depressed state canboth be constructed by expressing SITH-1, may be described as providinga result in good agreement with a clinical finding that antibodiesagainst SITH-1 are detected both from patients with manic-depressiveillness and from patients with depression.

7. Diagnosis Using SITH-1 as Marker

A study was made to see if diagnosis based on SITH-1 would also beuseful in diagnosing other diseases complicated by depression. Theresults are shown in FIG. 13.

Diagnosis based on the anti-SITH-1 antibody is quite specific to mooddisorders such as depression, manic-depressive illness, and chronicfatigue syndrome. However, as FIG. 13 shows, the same diagnosisexceptionally showed a high positive rate among patients with Crohn'sdisease. No positive outcome was shown by patients with ulcerativecolitis which was similar to Crohn's disease.

However, it is known that Crohn's disease is most frequently complicatedby “depressive symptoms”, and the anti-SITH-1 antibody positive personsshown in FIG. 13 are cases of Crohn's disease that were serious enoughto be complicated by depressive symptoms. The example underconsideration shows that even in such serious cases that patients withCrohn's disease which is a chronic disease classified as an autoimmunedisease also present with depressive symptoms, depression can bediagnosed using SITH-1 as a marker. In other words, testing with theanti-SITH-1 antibody may be considered to be “also useful in diagnosisof depression that is caused by other, non-psychiatric diseases.”

INDUSTRIAL APPLICABILITY

As described above, each of a gene and a protein of the presentinvention is a factor involved in latent infection with a herpesvirus.By using the gene and/or the protein as a marker, it is possible toobjectively determine whether or not a subject contracts a mentaldisorder. Therefore, the present invention not only provides benefits inacademic fields and basic research fields, but also has significance inclinical medicine fields. Therefore, the present invention is applicablein various aspects of industry.

1. A diagnosis method for diagnosing whether or not a non-human animalsubject has a mood disorder, comprising the steps of: (i) determiningwhether or not an antibody against a protein exists in the non-humananimal subject; and (ii) determining that the non-human animal subjecthas a mood disorder, wherein step (i) then determines that the antibodyexists at a diagnostic level in the non-human animal subject, saidprotein being selected from: (a) a protein having the amino sequence ofSEQ ID NO:1; and (b) a protein encoded by a gene comprising an openreading frame having the nucleotide sequence of SEQ ID NO:2, and whereinthe mood disorder is selected from the group consisting of depressionand manic-depressive illness.
 2. An animal model determination methodfor determining whether or not an animal subject is useful as an animalmodel of a mood disorder, comprising the steps of: (i) diagnosingwhether or not an animal subject has a mood disorder comprising thesteps of: (1) determining whether or not an antibody against a proteinexists in the non-human animal subject, said protein being selectedfrom: (a) a protein having the amino sequence of SEQ ID NO:1, and (b) aprotein encoded by a gene comprising an open reading frame having thenucleotide sequence of SEQ ID NO:2; and (2) determining that thenon-human animal subject has a mood disorder, wherein the mood disorderis selected from the group consisting of depression and manic-depressiveillness; and (ii) determining that the animal subject is useful as ananimal model of the mood disorder, in a case where the step (i)diagnoses that the animal subject has the mood disorder.
 3. An antibodydetection method for diagnosis of a mood disorder, comprising:detecting, in a sample obtained from a non-human subject, an antibodyagainst a protein, said protein being selected from: (a) a proteinhaving the amino acid sequence of SEQ ID NO: 1; and (b) a proteinencoded by a gene comprising an open reading frame having the nucleotidesequence of SEQ ID NO:2, wherein the mood disorder is selected from thegroup consisting of depression and manic-depressive illness.
 4. Adiagnosis method for diagnosing whether or not a non-human subject has amood disorder, comprising the steps of: (i) determining, in a sampleobtained from a non-human subject, a level of a protein, said proteinbeing selected from: (a) a protein having the amino acid sequence of SEQID NO: 1, and (b) a protein encoded by a gene comprising an open readingframe having the nucleotide sequence of SEQ ID NO:2; and (ii)determining that the non-human subject has a mood disorder, in a casewhere step (i) determines that the level of the protein is at adiagnostic level in the non-human subject, and wherein the mood disorderis selected from the group consisting of depression and manic-depressiveillness.